A glycated protein is generated non-enzymatically via a covalent bond between an amino group on a protein and the reducing terminal of a sugar, and is also referred to as an Amadori compound. In blood, glucose is bound to valine at the N-terminal of the β-chain of hemoglobin to generate glycated hemoglobin (glycohemoglobin; HbA1c). The abundance ratio of HbA1c to hemoglobin is higher in patients suffering from diabetes mellitus as compared with a normal healthy individual, and the concentration of HbA1c in blood is known to reflect the blood-sugar level during the past several weeks. Thus, concentration of HbA1c in blood is quite important in clinical tests for diagnosis of diabetes mellitus and in blood-sugar control of patients suffering from diabetes mellitus. The HbA1c concentration in blood may be measured using an enzyme having specificity to fructosyl valine or fructosyl valyl histidine.
Fructosyl amino acid oxidases have been isolated from various kinds of strains and it has been suggested that glycated proteins such as glycated albumin, HbA1c and fructosyl amino acids may be analyzed using such enzymes (JP A 61-268, 178; JP A 61-280, 297; JP A 03-155, 780; JP A 05-192, 193; JP A 07-289, 253; JP A 08-154, 672; JP A 2001-95598; JP A 2003-79386; JP A 2003-235585; Agric. Biol. Chem., 53(1), 103-110, 1989; Agric. Biol. Chem., 55(2), 333-338, 1991; J. Biol. Chem., 269(44), 27297-27301, 1994; Appl. Environ. Microbiol., 61(12), 4487-4489, 1995; Biosci. Biotech. Biochem., 59(3), 487-491, 1995; J. Biol. Chem., 270(1), 218-224, 1995; J. Biol. Chem., 271(51), 32803-32809, 1996; and J. Biol. Chem., 272(6), 3437-3443, 1997).
Fructosyl amino acid oxidase is a FAD-dependent enzyme which catalyzes a reaction where fructosyl amino acid is oxidized to generate 2-keto-D-glucose and the corresponding amino acid, while generating the reduced form of FAD (FADH2). FADH2 in turn transmits electrons to an electron acceptor and is converted to its oxidized form. In the presence of oxygen, FADH2 preferentially transmits electrons to the oxygen molecule rather than to artificial electron acceptors (also referred to as mediators or electron mediators). Thus, when a fructosyl amino acid is assayed by fructosyl amino acid oxidase with mediators, the assay results will be greatly affected by the dissolved oxygen level in the reaction system. This impact may be undesirable in clinical tests of blood samples by a point-of-care testing device utilizing an artificial electron acceptor. In some forms, it is desirable for the enzyme used for enzyme sensor strips employing artificial electron mediators to have low activity toward oxygen.
Accordingly, one non-limiting object of the present application is to provide an enzyme, such as a fructosyl amino acid oxidase, whose activity is less affected by the dissolved oxygen level. Further objects, embodiments, forms, features, advantages, aspects, and benefits shall become apparent from the following description and drawings.